Comparison between elementary flux modes analysis and 13C-metabolic fluxes measured in bacterial and plant cells

<p>Abstract</p> <p>Background</p> <p>¹³C metabolic flux analysis is one of the pertinent ways to compare two or more physiological states. From a more theoretical standpoint, the structural properties of metabolic networks can be analysed to explore feasible metabolic behaviours and to define the boundaries of steady state flux distributions. Elementary flux mode analysis is one of the most efficient methods for performing this analysis. In this context, recent approaches have tended to compare experimental flux measurements with topological network analysis.</p> <p>Results</p> <p>Metabolic networks describing the main pathways of central carbon metabolism were set up for a bacteria species (<it>Corynebacterium glutamicum</it>) and a plant species (<it>Brassica napus</it>) for which experimental flux maps were available. The structural properties of each network were then studied using the concept of elementary flux modes. To do this, coefficients of flux efficiency were calculated for each reaction within the networks by using selected sets of elementary flux modes. Then the relative differences - reflecting the change of substrate <it>i.e</it>. a sugar source for <it>C</it>. <it>glutamicum </it>and a nitrogen source for <it>B</it>. <it>napus </it>- of both flux efficiency and flux measured experimentally were compared. For both organisms, there is a clear relationship between these parameters, thus indicating that the network structure described by the elementary flux modes had captured a significant part of the metabolic activity in both biological systems. In <it>B</it>. <it>napus</it>, the extension of the elementary flux mode analysis to an enlarged metabolic network still resulted in a clear relationship between the change in the coefficients and that of the measured fluxes. Nevertheless, the limitations of the method to fit some particular fluxes are discussed.</p> <p>Conclusion</p> <p>This consistency between EFM analysis and experimental flux measurements, validated on two metabolic systems allows us to conclude that elementary flux mode analysis could be a useful tool to complement ¹³C metabolic flux analysis, by allowing the prediction of changes in internal fluxes before carbon labelling experiments.</p

Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves

Ammonium (NH4+)-based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH4+ provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic-driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data-constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non-aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.TP benefited from a cotutelle PhD (University of Bordeaux and University of the Basque Country) and thanks the University of the Basque Country (UPV/EHU, Spain) for his PhD grant during the execution of this work. This research was financially supported by the Basque Government (IT-932-16) and the Spanish Government (BIO2017-84035-R co-funded by Fondo Europeo para el Desarrollo Regional [FEDER]). Analytics were supported by MetaboHUB (ANR-11-INBS-0010) and PHENOME (ANR-11-INBS-0012) projects. Technical support was provided by Cedric Cassan, Ana Renovales and Mandy Bordas. The authors also thank SGIker (UPV/EHU, FEDER, EU) for the technical and human support provided

1H-NMR metabolomics: Profiling method for a rapid and efficient screening of transgenic plants

Metabolomics-based approaches are methods of choice for studying changes in fruit composition induced by  environmental or genetic modulation of biochemical pathways in the fruit. Owing to enzyme redundancy and  high plasticity of the metabolic network, transgenic alteration of the activity of the enzymes from the central metabolism very often results in only slight modifications of the fruit composition. In order to avoid costly and  time-consuming plant analysis, we used a fast and sensitive 1H-NMR-based metabolomic profiling technique  allowing discovery of slight metabolite variations in a large number of samples. Here, we describe the  screening of transgenic tomato plants in which two genes from the central metabolism, phosphoenolpyruvate  carboxylase (EC.3.4.1.1) and malate synthase (EC 2.3.3.9) were silenced by antisens and RNAi strategy.  1H-NMR metabolomic profiles of methanol-d4 D2O buffer extracts of tomato fruit flesh were acquired and  subjected to unsupervised multivariate statistical analysis. 1H-NMR spectra were binned into variable-size  spectral domains, making it possible to get an overall analysis of a large number of resonances, even in the  case of uncontrolled variation of the chemical shift. Principal component analysis was used to separate groups  of samples and to relate known and unknown metabolites to transgenic events. The screening of 100 samples,  from extraction to data mining, took 36 h. Thus, this procedure allows the rapid selection of metabolic  phenotypes of interest among about 30 transgenic lines.Key words: Metabolome, GMO, tomato, fruit, 1H-NMR profiling, screening

Trehalose synthase of Mycobacterium smegmatis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65905/1/j.1432-1033.2004.04365.x.pd

Sharks of the order Carcharhiniformes from the British Coniacian, Santonian and Campanian (Upper Cretaceous).

Bulk sampling of phosphate-rich horizons within the British Coniacian to Campanian (Upper Cretaceous) yielded very large samples of shark and ray teeth. All of these samples yielded teeth of diverse members of the Carcharhiniformes, which commonly dominate the fauna. The following species are recorded and described: Pseudoscyliorhinus reussi (Herman, 1977) comb. nov., Crassescyliorhinus germanicus (Herman, 1982) gen. nov., Scyliorhinus elongatus (Davis, 1887), Scyliorhinus brumarivulensis sp. nov., ? Palaeoscyllium sp., Prohaploblepharus riegrafi (Müller, 1989) gen. nov., ? Cretascyliorhinus sp., Scyliorhinidae inc. sedis 1, Scyliorhinidae inc. sedis 2, Pteroscyllium hermani sp. nov., Protoscyliorhinus sp., Leptocharias cretaceus sp. nov., Palaeogaleus havreensis Herman, 1977, Paratriakis subserratus sp. nov., Paratriakis tenuis sp. nov., Paratriakis sp. indet. and ? Loxodon sp. Taxa belonging to the families ?Proscylliidae, Leptochariidae, and Carcharhinidae are described from the Cretaceous for the first time. The evolutionary and palaeoecological implications of these newly recognised faunas are discussed

Combining Genomics, Metabolome Analysis, and Biochemical Modelling to Understand Metabolic Networks

Now that complete genome sequences are available for a variety of organisms, the elucidation of gene functions involved in metabolism necessarily includes a better understanding of cellular responses upon mutations on all levels of gene products, mRNA, proteins, and metabolites. Such progress is essential since the observable properties of organisms – the phenotypes – are produced by the genotype in juxtaposition with the environment. Whereas much has been done to make mRNA and protein profiling possible, considerably less effort has been put into profiling the end products of gene expression, metabolites. To date, analytical approaches have been aimed primarily at the accurate quantification of a number of pre-defined target metabolites, or at producing fingerprints of metabolic changes without individually determining metabolite identities. Neither of these approaches allows the formation of an in-depth understanding of the biochemical behaviour within metabolic networks. Yet, by carefully choosing protocols for sample preparation and analytical techniques, a number of chemically different classes of compounds can be quantified simultaneously to enable such understanding. In this review, the terms describing various metabolite-oriented approaches are given, and the differences among these approaches are outlined. Metabolite target analysis, metabolite profiling, metabolomics, and metabolic fingerprinting are considered. For each approach, a number of examples are given, and potential applications are discussed

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with beta-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical beta-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents

Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE

Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%

Deep EST profiling of developing fenugreek endosperm to investigate galactomannan biosynthesis and its regulation

Galactomannans are hemicellulosic polysaccharides composed of a (1 → 4)-linked β-D-mannan backbone substituted with single-unit (1 → 6)-α-linked D-galactosyl residues. Developing fenugreek (Trigonella foenum-graecum) seeds are known to accumulate large quantities of galactomannans in the endosperm, and were thus used here as a model system to better understand galactomannan biosynthesis and its regulation. We first verified the specific deposition of galactomannans in developing endosperms and determined that active accumulation occurred from 25 to 38 days post anthesis (DPA) under our growth conditions. We then examined the expression levels during seed development of ManS and GMGT, two genes encoding backbone and side chain synthetic enzymes. Based on transcript accumulation dynamics for ManS and GMGT, cDNA libraries were constructed using RNA isolated from endosperms at four ages corresponding to before, at the beginning of, and during active galactomannan deposition. DNA from these libraries was sequenced using the 454 sequencing technology to yield a total of 1.5 million expressed sequence tags (ESTs). Through analysis of the EST profiling data, we identified genes known to be involved in galactomannan biosynthesis, as well as new genes that may be involved in this process, and proposed a model for the flow of carbon from sucrose to galactomannans. Measurement of in vitro ManS and GMGT activities and analysis of sugar phosphate and nucleotide sugar levels in the endosperms of developing fenugreek seeds provided data consistent with this model. In vitro enzymatic assays also revealed that the ManS enzyme from fenugreek endosperm preferentially used GDP-mannose as the substrate for the backbone synthesis

Sharks and rays (chondrichthyes) from the Upper Cretaceous red beds of the south-central Pyrenees (Lleida, Spain): indices of an India-Eurasia connection

Six chondrichthyan taxa are recorded in Late Cretaceous (Maastrichtian) red beds of the south-central Pyrenees (Tremp Formation, Lleida, Spain), indicating marine influence in the palaeoenvironment. Two ray species found in Spain have been previously described in India. One ray species conforms to the Gondwanan genus 19dabatis and the endernic Indian species 19dabatis indicus Prasad and Cappetta, 1993 [Prasad, G.V.R., Cappetta, H., 1993. Late Cretaceous selachian from India and the age of the Deccan traps. Palaeontology 36 (1), 231-248.] The other ray from the Tremp Formation is also close to the Indian material assigned to Rhombodus sp. 2. The lirnited dispersal abilities of benthic rays points to a shallow trans-Tethyan connection between Eurasia and India at the end of the Cretaceous. The data increase the evidence of a biogeographical re1ationship between Eurasia and Gondwanan continents at the Cretaceous-Tertiary boundary